The invention relates generally to methods of making and using certain defined subsets of dendritic cells, more particularly, to methods of in vitro production of a subset of dendritic cells which produce large amounts of interferon.
The circulating component of the mammalian circulatory system comprises various cell types, including red and white blood cells of the erythroid and myeloid cell lineages. See, e.g., Rapaport (1987) Introduction to Hematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987) Blood: Textbook of Hematology, Little, Brown and Co., Boston, Mass.; and Paul (ed. 1993) Fundamental Immunology (3d ed.) Raven Press, N.Y.
Dendritic cells (DCs) are the most potent of antigen-presenting cells. See, e.g., Paul (ed. 1993) Fundamental Immunology 3d ed., Raven Press, NY. Antigen presentation refers to the cellular events in which a proteinaceous antigen is taken up, processed by antigen presenting cells (APC), and then recognized to initiate an immune response. The most active antigen presenting cells have been characterized as the macrophages (which are direct developmental products from monocytes), dendritic cells, and certain B cells. DCs are highly responsive to inflammatory stimuli such as bacterial lipopolysaccharides (LPS) and cytokines such as tumor necrosis factor alpha (TNFxcex1). The presence of cytokines and LPS can induce a series of phenotypic and functional changes in DC that are collectively referred to as maturation. See, e.g., Banchereau and Schmitt Dendritic Cells in Fundamental and Clinical Immunology Plenum Press, NY.
Dendritic cells can be classified into various categories, including: interstitial dendritic cells of the heart, kidney, gut, and lung; Langerhans cells in the skin and mucous membranes; interdigitating dendritic cells in the thymic medulla and secondary lymphoid tissue; and blood and lymph dendritic cells. Although dendritic cells in each of these compartments are CD45+ leukocytes that apparently arise from bone marrow, they may exhibit differences that relate to maturation state and microenvironment. Maturational changes in DCs include, e.g., silencing of antigen uptake by endocytosis, upregulation of surface molecules related to T cell activation, and active production of a number of cytokines including TNFxcex1 and IL-12. Upon local accumulation of TNFxcex1, DCs migrate to the T cell areas of secondary lymphoid organs to activate antigen specific T cells.
Many factors have been identified which influence the differentiation process of precursor cells, or regulate the physiology or migration properties of specific cell types. See, e.g., Mire-Sluis and Thorpe (1998) Cytokines Academic Press, San Diego; Thomson (ed. 1998) The Cytokine Handbook (3d ed.) Academic Press, San Diego; Metcalf and Nicola (1995) The Hematopoietic Colony Stimulating Factors Cambridge University Press; and Aggarwal and Gutterman (1991) Human Cytokines Blackwell. These factors provide yet unrecognized biological activities, e.g., on different untested cell types.
However, dendritic cells are poorly characterized, both in terms of responses to soluble factors, and many of their functions and mechanisms of action. The absence of knowledge about the physiological properties and responses of these cells limits their understanding. Thus, medical conditions where regulation, development, or physiology of dendritic cells is unusual remain unmanageable. The present invention addresses these issues.
The present invention is based, in part, upon the surprising discovery of conditions which result in large numbers of viable type I IFN producing cells, or pDC2 cells. The invention provides methods comprising contacting CD34++CD45RAxe2x88x92 early haematopoietic progenitor cells with an effective amount of FLT3 ligand ex vivo, thereby inducing differentiation of the cells to IFN producing DC. Typically, the effective amount is at least 70 ng/ml; the contacting is for at least 15 days; the IFN producing DC produce at least 5000 pg IFN per 20,000 cells over 24 h after viral stimulation; the early progenitor cells expand at least about 10 fold; and/or the IFN producing cells number at least 2.5 million. In a preferred embodiment, the contacting is with TPO, and the early progenitor cells expand at least 30 fold. In other embodiments, the early progenitor cells expand at least 100 fold; or after the expansion, at least 3% of the resulting cell culture is IFN producing DC; or the IFN producing DC accumulate in 24 h at least 40,000 pg IFN per 20,000 cells after viral stimulation.
In other embodiments, the invention provides methods of producing IPC comprising contacting IPC precursors with an effective amount of a combination of both FLT3 Ligand and TPO. Preferably, the contacting is for at least 13 days; the precursors are CD34++CD45RAxe2x88x92 early haematopoietic progenitor cells; the IPC accumulate in 24 h at least 5000 pg IFN per 20,000 IPC after viral stimulation; and/or the IPC number at least 1xc3x97107 cells. Typically, the contacting is ex vivo.
In yet another embodiment, the invention provides populations of at least 3xc3x97106 viable IPC derived from a single individual, e.g., at least 7, 10, or 15xc3x97106 cells. Preferably, cells are cultured in the presence of both FLT3 Ligand and TPO to produce the IPC, e.g., in vitro for at least 14 days where the FLT3 ligand is at least 70 ng/ml; and/or the TPO is at least 70 ng/ml. Typically, the IPC are CD34xe2x88x92CD45RA++CD4+IL-3Rxcex1++ cells.
Outline
I. General
A. IPC
B. Developmental pathway
II. Producing IPC
A. FLT3 Ligand
B. thrombopoietin (TPO)
C. Other Molecules
III. Uses
I. General
Natural Interferon-xcex1 producing cells (IPC) are key effector cells in anti-viral innate immunity. These cells produce up to 1000 times more IFN-xcex1 than other blood cell types in response to viral stimulation. IPCs also have the capacity to become dendritic cells, which are key antigen presenting cells in the induction of T cell mediate immune responses.
Upon viral stimulation, the natural IFN-xcex1/xcex2 producing cells (IPCs, also known as pre-DC2) in human blood and peripheral lymphoid tissues rapidly produce very large amounts of IFN-xcex1/xcex2. After performing this innate anti-viral immune response, IPCs can differentiate into dendritic cells and strongly stimulate T cell mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, the developmental pathway has been mapped herein to pre-DC2/IPCs from CD34+ heamatopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages have been identified, including CD34++CD45RAxe2x88x92 early progenitor cells, CD34++CD45RA+ late progenitor cells, CD34+CD45RA++CD4+IL-3Rxcex1++pro-DC2, and CD34xe2x88x92CD45RA++CD4+IL-3Rxcex1++ pre-DC2/IPCs. Pro-DC2s already have acquired the capacity to produce large amounts of IFN-xcex1/xcex2 upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40-Ligand. The expression of pre-T cell receptor (TCR) alpha chain mRNA by both pro-DC2 and pre-DC2 supports the lymphoid origin of the pre-DC2/IPC lineage. CD34++CD45RAxe2x88x92 early progenitor cells did not have the capacity to produce large amounts of IFN-xcex1/xcex2 in response to viral stimulation, however they can be induced to undergo clonal expansion and differentiation into IPCs/Pre-DC2 in culture with FLT3-Ligand.
Dendritic cells (DCs) represent heterogeneous populations of heamatopoietic-derived cells that display potent ability to induce primary T cell activation, polarization, and in certain circumstances tolerance. See Sousa, et al. (1999) Curr. Op. Immunol. 11:392-399; Sallusto and Lanzavecchia (1999) J. Exp. Med. 189:611-614; Banchereau and Steinman (1998) Nature 392:245-252; Cella, et al. (1997) Curr. Opin. Immunol. 9:10-16; and Steinman (1991) Annu. Rev. Immunol. 9:271-296. The distinct capacity of DCs to induce immunity versus tolerance or Th1 versus Th2 responses depends on their maturation stage (Cella, et al. (1997) Curr. Opin. Immunol. 9:10-16; and Kalinski, et al. (1999) Immunol. Today 20:561-567), signals that induce or inhibit DC maturation (Cella, et al. (1997) Curr. Opin. Immunol. 9:10-16; and Kalinski, et al. (1999) Immunol. Today 20:561-567; d""Ostiani, et al. (2000) J. Exp. Med. 191:1661-1674), as well as the lineage origin of DCs (Pulendran, et al. (1999) Proc. Nat""l Acad. Sci. USA 96:1036-1041; Reis e Sousa, et al. (1999) Curr. Opin. Immunol. 11:392-399; Maldonado-Lopez, et al. (1999) J. Exp. Med. 189:587-592; Arpinati, et al. (2000) Blood 95:2484-2490; Liu and Blom (2000) Blood 95:2482-2483; and Shortman (2000) Immunol. Cell Biol. 78:161-165). A lymphoid DC developmental pathway was suggested by the finding that mouse thymic lymphoid precursors can give rise to both T cells and CD8+CD11bxe2x88x92 DCs. Ardavin, et al. (1993) Nature 362:761-763; and Shortman, et al. (1998) Immunol. Rev 165:39-46. In addition, a well-established myeloid DC pathway giving rise to CD8xe2x88x92CD11b+ DCs has been defined. Inaba, et al. (1992) J. Exp. Med. 176:1693-1702; Inaba, et al. (1993) Proc. Nat""l Acad. Sci. USA 90:3038-2042; and Young and Steinman (1996) Stem Cells 14:376-287. Recent studies suggest that CD8+CD11 bxe2x88x92 lymphoid DCs and CD8xe2x88x92CD11b+ myeloid DCs may have different functions in T cell activation/tolerance or Th1/Th2 differentiation. Pulendran, et al. (1999) Proc. Nat""l Acad. Sci. USA 96:1036-1041; Maldonado-Lopez, et al. (1999) J. Exp. Med. 189:587-592; Suss and Shortman (1996) J. Exp. Med. 183:1789-1796; Kronin, et al. (1997) Int. Immunol. 9:1061-1064; Stumbles, et al. (1998) J. Exp. Med. 188:2019-2031; Ohteki, et al. (1999) J. Exp. Med. 189:1981-1986; Thomson, et al. (1999) J. Leukoc. Biol. 66:322-330; Iwasaki and Kelsall (1999) J. Exp. Med. 190:229-239; and Khanna, et al. (2000) J. Immunol. 164:1346-1354.
In humans, two distinct populations of dendritic cell precursors have been identified in the blood. Monocytes (pre-DC1), which belong to the myeloid lineage, differentiate into immature DC1 after 5 days of culture in granulocyte colony-stimulating factor (GM-CSF) and IL-4. Sallusto and Lanzavecchia (1994) J. Exp. Med. 179:1109-1118; and Romani, et al. (1994) J. Exp. Med. 180:83-93. Upon CD40-Ligand activation, immature myeloid DC1 undergo maturation and produce large amounts of IL-12. Cella, et al. (1996) J. Exp. Med. 184:747-752; and Koch, et al. (1996) J. Exp. Med. 184:741-746. The mature DC1 induced by CD40-Ligand are able to polarize naive CD4+ T cells into Th1 cells. Rissoan, et al. (1999) Science 283:1183-1186. The second type of DC precursor cells, pre-DC2 (previously known as plasmacytoid T/monocytes) are characterized by a unique surface phenotype (CD4+IL-3Rxcex1++CD45RA+HLA-DR+ lineage markersxe2x88x92 and CD11cxe2x88x92), and at the ultrastructural level resemble immunoglobulin-secreting plasma cells. Grouard, et al. (1997) J. Exp. Med. 185:1101-1111; and Facchetti, et al. (1999) Histopathology 35:88-89. Several lines of evidence suggest that pre-DC2s are of lymphoid origin: i) pre-DC2 lack expression of the myeloid antigens CD11c, CD13, CD33, and mannose receptor (Grouard, et al. (1997) J. Exp. Med. 185:1101-1111; and Res, et al. (1999) Blood 94:2647-2657), ii) pre-DC2 isolated from the thymus, express the lymphoid markers CD2, CD5, and CD7 (Res, et al. (1999) Blood 94:2647-2657), iii) pre-DC2 have little phagocytic activity (Grouard, et al. (1997) J. Exp. Med. 185:1101-1111), iv) pre-DC2 do not differentiate into macrophages following culture with GM-CSF and macrophage-colony stimulating factor (M-CSF) (Grouard, et al. (1997) J. Exp. Med. 185:1101-1111), v) pre-DC2 express pre-TCR alpha transcripts (Res, et al. (1999) Blood 94:2647-2657; and Bruno, et al. (1997) J. Exp. Med. 185:875-884), and vi) development of pre-DC2, T and B cells, but not myeloid DC is blocked by ectopic expression of inhibitor of DNA binding (Id)2 or Id3. Pre-DC2 differentiate into immature DC2 when cultured with monocyte conditional medium (O""Doherty, et al. (1994) Immunology 82:487-493), IL-3 (Rissoan, et al. (1999) Science 283:1183-1186; Grouard, et al. (1997) J. Exp. Med. 185:1101-1111; and Olweus, et al. (1997) Proc. Nat""l Acad. Sci. USA 94:12551-12556), IFN-xcex1/xcex2 and tumor necrosis factor (TNF)-xcex1 or viruses, like Herpes Simplex Virus or Influenza virus (Kadowaki, et al. (2000) J. Exp. Med. 192:219-226). Upon CD40-Ligand activation, immature DC2 undergo maturation (Grouard, et al. (1997) J. Exp. Med. 185:1101-1111), but produce only low levels of IL-12 (Rissoan, et al. (1999) Science 283:1183-1186). Mature DC2 are able to polarize naxc3xafve CD4+ T cells into a Th2 phenotype (Arpinati, et al. (2000) Blood 95:2484-2490; and Rissoan, et al. (1999) Science 283:1183-1186). Recent studies showed that the pre-DC2 are the elusive natural interferon producing cells (IPC), capable of producing high amounts of IFN-xcex1/xcex2 upon viral stimulation (Siegal, et al. (1999) Science 284:1835-1837; and Cella, et al. (1999) Nature Med. 5:919-923). Taken together, pre-DC2/IPCs represent a unique heamatopoietic lineage, capable of performing crucial functions both in innate and in adapted immunity.
The pathway underlying the development of pre-DC2/IPC from CD34+ heamatopoietic stem cells has not been elucidated. Caux, et al. (1997) Blood 90:1458-1470 showed that cord blood CD34+ heamatopoietic progenitor cells cultured in GM-CSF, stem cell factor (SCF), and TNF-xcex1 differentiate along two DC pathways: i) the Langerhans cell (LC) pathway, in which intermediate CD14xe2x88x92CD1a+ DC precursors differentiated into LCs characterized by the expression of CD1a, Birbeck granules, the Lag antigen, and E cadherin; and ii) the dermal DC pathway, in which intermediate CD14+CD1axe2x88x92 DC precursors differentiate into dermal DCs characterized by the expression of CD1a, CD9, CD68, CD2, and factor XIIIa (Caux, et al. (1996) J. Exp. Med. 184:695-706). Recently, a common human lymphoid progenitor (CLP) in the bone marrow was described that expresses both CD45RA and CD10. Galy, et al. (1995) Immunity 3:459-473. These cells develop into T, B, NK cells, and DC, but not into erythroid, megakaryocytic, and myeloid cells. In these experiments, exclusively CD1a+ LCs were generated with a cocktail of 9 cytokines (IL-1, IL-3, IL-6, IL-7, SCF, GM-CSF, TNF, erythropoietin (EPO), and FLT3-Ligand). Of these cytokines, the heamatopoietic growth factor FLT3-Ligand has been shown to play an important role in the proliferation, survival, and differentiation of early murine and human heamatopoietic precursor cells. Zeigler, et al. (1994) Blood 84:2422-2430; and Shurin, et al. (1998) Cytokine Growth Factor Rev. 9:37-48. Interestingly, volunteer donors injected with FLT3-Ligand had a 13-fold increase in pre-DC2 number and a 48-fold increase in CD11c+ myeloid DC number in the blood stream. Pulendran, et al. (2000) J. Immunol. 165:566-572. Consistent with this finding, injection of mice with human FLT3-Ligand led to dramatically increased numbers of both myeloid and lymphoid DC not only in the peripheral blood, but also in the bone marrow, thymus, and secondary lymphoid tissues. Pulendran, et al. (1999) Proc. Nat""l Acad. Sci. USA 96:1036-1041; Maldonado-Lopez, et al. (1999) J. Exp. Med. 189:587-592; Maraskovsky, et al. (1996) J. Exp. Med. 184:1953-1962; and Pulendran, et al. (1997) J. Immunol. 159:2222-2231. Two recent reports revealed a 5 to 6-fold increase in pre-DC2/IPC numbers in the blood of granulocyte-colony stimulating factor (G-CSF) treated donors. Arpinati, et al. (2000) Blood 95:2484-2490; and Pulendran, et al. (2000) J. Immunol. 165:566-572. It is not clear from these studies, however, whether FLT3-Ligand and G-CSF enhance the differentiation of pre-DC2 from heamatopoietic progenitor cells, or promote the migration of pre-DC2 from bone marrow into blood.
The aims of the current studies are: i) to trace the developmental pathway of pre-DC2/IPCs from CD34++ heamatopoietic progenitor cells; and ii) to identify the stimuli that can induce CD34++ heamatopoietic progenitor cells to differentiate into pre-DC2/IPCs.
Herein is described the identification of CD34-expressing precursors of pre-DC2/IPCs from human fetal tissues and cord blood. In addition, the generation of pre-DC2/IPCs from early heamatopoietic stem cells in vitro cultures is described.
II. Producing IPC
Various aspects of the IPCs have been described, e.g., in Kadowaki, et al. (2000) J. Expt""l Med. 19:219-226; Rissoan, et al. (1999) Science 283:1183-1186; and Grouard, et al. (1997) J. Exp. Med. 185:1101-1111. The IFN-xcex1 production assays are described below, but can be immunoassays after viral induction. An appropriate virus is selected, e.g., HSV-1, KOS strain, attenuated by xcex3-irradiation, to infect the cell cultures. Typical multiplicity of infection numbers are 1, 3, 5, 7, 10, 13, 17, or 20 pfu/cell. The amount of IFN-xcex1 produced is determined after accumulation for a defined period of time, e.g., 12, 18, 24, or 36 h. The amounts of IFN produced will vary according to individual and other parameters, but will be at least about 5000 pg IFN per 20,000 cells accumulated over 24 h, but will preferably be more, e.g., 10, 20, 30, 40, 50, 60, or 70 thousand, or more.
DC precurser populations may originate from various sources, e.g., fetal liver, cord blood, bone marrow, or G-CSF mobilized blood. From adults, bone marrow derived precursors may be derived from biopsy or fresh cadaver samples. G-CSF mobilization may use appropriate amounts of cytokine, without or with FLT3 ligand. See, e.g., Mire-Sluis and Thorpe (1998) Cytokines Academic Press, San Diego; Thomson (ed. 1998) The Cytokine Handbook (3d ed.) Academic Press, San Diego; Metcalf and Nicola (1995) The Hematopoietic Colony Stimulating Factors Cambridge University Press; Aggarwal and Gutterman (1991) Human Cytokines Blackwell; WO97/12633; and WO99/26639. After mobilization, precursors may be isolated, e.g., by leukophoresis, and used in methods analogous to those described.
Isolation of these IPCs is inefficient from natural sources due, in part, to their rarity. Though small numbers can be isolated from natural sources, they are very fragile and not easily culturable to provide viable cells. Moreover, there are limitations on amounts of material that can be harvested from a single individual. The cells cannot be stored to remain viable to allow multiple isolations from a single individual to be pooled. However, the present methods allow for the proliferation and expansion of precursors to provide larger numbers of the cells from a single individual. In vitro methods using FLT3 Ligand are provided, and combinations of FLT3 Ligand with TPO provide even larger numbers of IPCs.
DC precursors are cultured under appropriate conditions, e.g., in various cytokines to induce proliferation and sustain development. Many combinations of cytokines among FLT-3 Ligand, SCF, IL-7, IL-3, G-CSF, and GM-CSF can inhibit development, and/or fail to support cell proliferation. FLT-3 Ligand sustains the combination of both proliferation and differentation. In combination with thrombopoietin (TPO; Shimomura, et al. (2000) Int. J. Hematol. 71:33-39; and Qiu, et al. (1999) J. Hematother. Stem Cell Res. 8:609-618), FLT3 Ligand has a dramatic effect on both proliferation and maintenance of differentiation of DC populations. The length of time and amounts of cytokines for such proliferation and development are routinely optimized. Under the conditions described herein, the time of contacting precursors with cytokine is from at least about 7, 10, 13, 15, 17, 19, 21, 23, 25, or 27 days. The appearance of the pDC2 cells occurs over those time periods, and the proportion of cells is in the range from 4, 6, 8, or 10% or more. The amounts of cytokine used had been optimized around, for IL-3 10 ng/ml; for GM-CSF 800 U/ml; for FLT-3 Ligand 100 ng/ml; for TPO 100 ng/ml; for SCF 10 ng/ml; for IL-7 10 ng/ml; and for G-CSF 5 ng/ml. However, these may be titrated, and should have similar effects to the respective cytokine at amounts of, e.g., 30%, 50%, 70%, 90%, 110%, 130%, etc. The appearance of the pDC2, under the described conditions, typically begins at about 11 days and increases at 13, 15, 17, 19, 21, and 23 days. The numbers of the cells seem to peak at about 25-28 days. Viability of cells may decrease thereafter, or the cells may further differentiate or lose their differentation markers.
The conditions evaluated herein are directed primarily to in vitro cultures, but the periods for in vivo treatment should be comparable or perhaps even shorter periods of time, e.g., by 50%, 60%, 70%, 80%, 90%, or so.
Conversely, it would be expected that the differentiation of pDC2 may be blocked by blocking the signals mediated by the indicated cytokines. Thus, antagonists of FLT3 Ligand and/or TPO may be administered in critical windows or longer term to block the differentiation of these cells. Thus, in circumstances where IPC normally produce IFN, antagonists may lower the systemic or local IFN levels.
Recombinant or other sources of the cytokines are known, and can be administered in culture or in vivo as appropriate. Recombinant protein can be expressed and purified in eukaryotic or prokaryotic cells as described, e.g., in Coligan, et al. (eds. 1995 and periodic supplements) Current Protocols in Protein Science John Wiley and Sons, New York, N.Y.; and Ausubel, et al (eds. 1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York, N.Y.
Alternatively, antagonists are available, e.g., antibodies to ligands, soluble receptors, mutein antagonists, etc. Naturally folded or denatured material, perhaps expressed on cell surfaces, can be used, as appropriate, for producing antibodies. Either monoclonal or polyclonal antibodies may be generated, e.g., for subsequent use in immunopurification methods.
III. Uses
IPC will be important in a number of therapeutic and research applications. See, e.g., Kadowaki, et al. (2000) J. Expt""l Med. 192:219-226; and Liu and Blom (2000) Blood 95:2482-2483. They will be used in cellular therapy for viral infections and diseases, e.g., HIV or hepatitis. The cells will produce natural interferons, and can substitute for administration of the interferons in treatment of medical conditions. The cells may be produced in vivo or ex vivo according to methods described. These methods also provide means to isolate large quantities of pDC2 cells, which will allow for further study and characterization. These cells will be useful in studying the molecular mechanisms regulating IFN-xcex1 production.
In contrast, there will be circumstances where the generation of pDC2 cell types may be counter indicated. Prevention of such differentation may be effected by blockage of signaling mediated by the respective differentiation factors. This may take the forms of mutein antagonists, antibody antagonists, receptor antibody antagonists, soluble receptor constructs, small molecule antagonists, etc. Such may be indicated where high levels of IFN are deleterious, e.g., in autoimmune contexts such as lupus (see, e.g., Vallin, et al. (1999) Clin. Exp. Immunol. 115:196-202; Schilling, et al. (1991) Cancer 68:1536-1537), or in tumor contexts.
So, the present invention provides means to produce and purify desired dendritic cell subsets, or alternatively to block such. Alternatively, labeling can be used to FACS sort cells which specifically express these markers. Populations of substantially homogeneous IPCs will have important utility in research or therapeutic environments.
Effects on various cell types may be indirect, as well as direct. A statistically significant change in the numbers of cells will typically be at least about 10%, preferably 20%, 30%, 50%, 70%, 90%, or more. Effects of greater than 100%, e.g., 130%, 150%, 2xc3x97, 3xc3x97, 5xc3x97, etc., will often be desired. The effects may be specific in numbers or proportions of the various cell subpopulations.
The present invention will be useful in the treatment of medical conditions or diseases associated with innate or viral immunity. See, e.g., Frank, et al. (eds. 1995) Samter""s Immunologic Diseases, 5th Ed., vols. I-II, Little, Brown and Co., Boston, Mass.
The cells or cytokines described may be combined with other treatments of the medical conditions described herein, e.g., an antibiotic, antifungal, antiviral, immune suppressive therapeutic, immune adjuvant, analgesic, anti-inflammatory drug, growth factor, cytokine, vasodilator, or vasoconstrictor. See, e.g, the Physician""s Desk Reference, both prescription and non-prescription compendiums. Preferred combination therapies include the cells or reagents with various anti-infective agents.
Standard immunological techniques are described, e.g., in Hertzenberg, et al. (eds. 1996) Weir""s Handbook of Experimental Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.; and Methods in Enzymology volumes 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163. These will allow use of the reagents for purifying cell subpopulations, etc.
To prepare pharmaceutical or sterile compositions including, e.g., TPO, the material is admixed with a pharmaceutically acceptable carrier or excipient which is preferably inert. Preparation of such pharmaceutical compositions is known in the art, see, e.g., Remington""s Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Typically, therapeutic compositions are sterile. Alternatively, FLT3 Ligand and/or TPO antagonist compositions can be prepared.
Agonists, e.g., natural ligand, or antagonists, e.g., antibodies or binding compositions, are normally administered parenterally, preferably intravenously. Since such protein or peptide antagonists may be immunogenic they are preferably administered slowly, either by a conventional IV administration set or from a subcutaneous depot, e.g. as taught by Tomasi, et al., U.S. Pat. No. 4,732,863.
When administered parenterally the therapeutics will typically be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic and nontherapeutic. The antagonist may be administered in aqueous vehicles such as water, saline, or buffered vehicles with or without various additives and/or diluting agents. Alternatively, a suspension, such as a zinc suspension, can be prepared to include the peptide. Such a suspension can be useful for subcutaneous (SQ), intradermal (ID), or intramuscular (IM) injection. The proportion of therapeutic entity and additive can be varied over a broad range so long as both are present in effective combination amounts. The therapeutic is preferably formulated in purified form substantially free of aggregates, other proteins, endotoxins, and the like, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20 mg/ml. Preferably, the endotoxin levels are less than 2.5 EU/ml. See, e.g., Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications 2d ed., Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets 2d ed., Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY; Fodor, et al. (1991) Science 251:767-773; Coligan (ed.) Current Protocols in Immunology; Hood, et al. Immunology Benjamin/Cummings; Paul (ed. 1997) Fundamental Immunology 4th ed., Academic Press; Parce, et al. (1989) Science 246:243-247; Owicki, et al. (1990) Proc. Nat""l Acad. Sci. USA 87:4007-4011; and Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.
Selecting an administration regimen for a therapeutic agonist or antagonist depends on several factors, including the serum or tissue turnover rate of the therapeutic, the immunogenicity of the therapeutic, or the accessibility of the target cells. Preferably, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of therapeutic delivered depends in part on the particular agonist or antagonist and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies is found in the literature on therapeutic uses, e.g. Bach et al., chapter 22, in Ferrone, et al. (eds. 1985) Handbook of Monoclonal Antibodies Noges Publications, Park Ridge, N.J.; and Russell, pgs. 303-357, and Smith, et al., pgs. 365-389, in Haber, et al. (eds. 1977) Antibodies in Human Diagnosis and Therapy Raven Press, New York, N.Y.
Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Numbers of pDC2 cells in defined samples might be important indicators of when an effective dose is reached. Preferably, an antibody or binding composition thereof that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing a humoral response to the reagent.
The total weekly dose ranges for antibodies or fragments thereof, which specifically bind to cytokine, range generally from about 1 ng, more generally from about 10 ng, typically from about 100 ng; more typically from about 1 xcexcg, more typically from about 10 xcexcg, preferably from about 100 xcexcg, and more preferably from about 1 mg per kilogram body weight. Although higher amounts may be more efficacious, the lower doses typically will have fewer adverse effects. Generally the range will be less than 100 mg, preferably less than about 50 mg, and more preferably less than about 25 mg per kilogram body weight.
The weekly dose ranges for antagonists, e.g., antibody, binding fragments, range from about 10 xcexcg, preferably at least about 50 xcexcg, and more preferably at least about 100 xcexcg per kilogram of body weight. Generally, the range will be less than about 1000 xcexcg, preferably less than about 500 xcexcg, and more preferably less than about 100 xcexcg per kilogram of body weight. Dosages are on a schedule which effects the desired treatment and can be periodic over shorter or longer term. In general, ranges will be from at least about 10 xcexcg to about 50 mg, preferably about 100 xcexcg to about 10 mg per kilogram body weight.
Other antagonists of the ligands, e.g., muteins, are also contemplated. Hourly dose ranges for muteins range from at least about 10 xcexcg, generally at least about 50 xcexcg, typically at least about 100 xcexcg, and preferably at least 500 xcexcg per hour. Generally the dosage will be less than about 100 mg, typically less than about 30 mg, preferably less than about 10 mg, and more preferably less than about 6 mg per hour. General ranges will be from at least about 1 xcexcg to about 1000 xcexcg, preferably about 10 xcexcg to about 500 xcexcg per hour.
The phrase xe2x80x9ceffective amountxe2x80x9d means an amount sufficient to effect a desired response, or to ameliorate a symptom or sign of the target condition. Typical mammalian hosts will include mice, rats, cats, dogs, and primates, including humans. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route, and dose of administration and the severity of side affects. Preferably, the effect will result in a change in quantitation of at least about 10%, preferably at least 20%, 30%, 50%, 70%, or even 90% or more. When in combination, an effective amount is in ratio to a combination of components and the effect is not necessarily limited to individual components alone.
An effective amount of therapeutic will modulate the symptoms typically by at least about 10%; usually by at least about 20%; preferably at least about 30%; or more preferably at least about 50%. Such will result in, e.g., statistically significant and quantifiable changes in the numbers of cells being affected. This may be an increase or decrease in the numbers of target cells appearing within a time period or target area.
The present invention provides reagents which will find use in therapeutic applications as described elsewhere herein. See, e.g., Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck and Co., Rahway, N.J.; Thorn, et al. Harrison""s Principles of Internal Medicine, McGraw-Hill, N.Y.; Gilman, et al. (eds. 1990) Goodman and Gilman""s: The Pharmacological Bases of Therapeutics. 8th Ed., Pergamon Press; Remington""s Pharmaceutical Sciences. 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Langer (1990) Science 249:1527-1533; and Merck Index, Merck and Co., Rahway, N.J.
Antibodies to marker proteins may be used for the identification or sorting of cell populations expressing those markers. Methods to sort such populations are well known in the art, see, e.g., Melamed, et al. (1990) Flow Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.
Moreover, antisense nucleic acids may be used. For example, antisense polynucleotides against the ligand encoding nucleic acids may function in a manner like ligand antagonists, and antisense against the receptor may function like receptor antagonists. Thus, it may be possible to block the signaling through the pathway with antisense nucleic acids. Conversely, nucleic acids for the receptor may serve as agonists, increasing the numbers of receptor on the cell, thereby increasing cell sensitivity to ligand, and perhaps blocking the normal apoptotic signal described.